INTRODUCTION:

The rapid development of protein-based biopharmaceuticals in recent years has opened up new avenues of research related to protein instabilities (aggregation and degradation).^[1,2] Protein-based biopharmaceuticals are used in the treatment of a number of medical conditions including auto‐immune diseases, cancer, mental disorder, hypertension, and certain cardiovascular, diabetes, rheumatoid arthritis, hepatitis and metabolic disorders. ^[3-5] They are extracted or synthesized from biological sources including microbes, animals, plants or humans. They are globular proteins, with specific spatial conformations, that enable them to perform their biological functions. Protein-based biopharmaceuticals are often prone to aggregation comprised of up to several thousand interconnected protein subunits. Non-native irreversible aggregation of proteins affects multiple steps in the production, including expression, purification, freezing, handling and long-term storage^{. [6, 7]} Protein-based biopharmaceuticals are currently available as liquid and/or lyophilized products. ^[8] Liquid products are preferred as they are cheaper to produce and easier to use than lyophilized products. However, for some protein-based biopharmaceuticals, chemical or physical stability aspects are difficult to control adequately in the liquid state.One such physical instability is the high tendency of protein molecules to aggregate under a wide range of processing and storage conditions. Protein aggregates are assemblies of native or partially denatured (unfolded) protein molecules that are considered product-related degradation products. The presence of protein aggregates can affect the protein drug efficacy, ^[9-11] but a primary concern is that they may enhance the product’s immunogenicity. ^[12] Aggregation is therefore a fundamental cause for assessing the quality of protein drugs. The initial protein aggregates are soluble oligomers but gradually become insoluble as they exceed certain size and solubility limits resulting in precipitation of different forms such as amorphous precipitates (disordered protein aggregates) or fibrils (ordered protein aggregates), depending on the protein structures and experimental conditions. ^[13] Protein aggregation follows two main pathways. The first is the aggregation of native protein conformations (colloidal instability) and the second is the aggregation of partially denatured (non-native) proteins. ^{[9, 14]} Both aggregation pathways may occur for a single protein^{. [15]}In order to minimize or prevent protein aggregation, both of these two pathways should be targeted. ^[16] Among factors that affect protein aggregation are protein structure-related and formulation or process-related that include surface adsorption, temperature, pH and excipients. Both factors are interconnected; for example, one protein molecule might aggregate under one set of formulation or process-related factors, while another protein is stable under the same formulation or process-related factors. Manipulation of formulation or process-related factors is usually the first choice for achieving product stability. This approach involves changing the formulation or process-related factors. It consists of two steps; enhancing the conformational stability of the native proteins and inducing intermolecular repulsive forces of the native proteins. ^[14]If a protein-based biopharmaceutical candidate has a very high tendency to aggregate, changing the formulation or process-related factors may not be sufficient to prevent aggregation. In this case, another approach can be attempted, which is the structural modification of the protein either genetically or chemically. This approach may have a major limitation of the possible reduction or even complete loss in protein activity. The identification and quantification of protein aggregation can be determined using size-exclusion chromatography (SEC) with either UV or light scattering detection and is considered as one of the quality control/quality assurance test. Another method used for aggregate analysis is polyacrylamide gel electrophoresis (PAGE). ^[17] The aggregation of protein drugs not only decreases their overall activity, but also elicits immunological reactions. ^{[18, 19]}One of the major factors that may influence the stability of protein-based biopharmaceuticals is temperature. Most often, extracted proteins are stored for an extended period at low temperatures to test their abilities in maintaining their activities and original structural integrities. Usually, proteins are best stored at 2-8°C. Storage at room temperature often leads to the degradation of most protein drugs. Furthermore, proteins formulated as parenteral sustained release products may encountered in-vivo instability problems that may result in the formation of protein aggregation. This may induce an immune response in patients treated with sustained release products of protein drugs. ^[20] A number of approaches can be applied to enhance the stability of protein-based biopharmaceuticals. The first approach is to change the amino acid sequences in the protein structure. ^[21] The second one is to optimize the formulation of the protein-based biopharmaceuticals. ^[22,
23]

A number of polymers have been observed to increase the stability of protein-based biopharmaceuticals; these include thermo-sensitive polymers. ^[18] or the use of nontoxic nanostructured materials. ^[24] This review highlights the instability of protein-based biopharmaceuticals, their long-term storage stability prediction as well as the clinical consequences resulting from such instability.

Aggregation pathways:

In aqueous solutions, the native protein conformation is spontaneously achieved, because the burial of hydrophobic moieties in the protein core is energetically favorable, that acts as the driving force for protein folding. ^[25] Although most of the protein core is composed of hydrophobic moieties, structures in the Protein Data Bank (PDB) show that acidic and basic side chains are often buried in the protein core, in addition to the presence of acidic and basic side chains on the globular protein surface. ^[26] Various types of weak intramolecular forces such as hydrogen bonds, salt bridges and weak Vander Waal’s bonds also contribute to the conformational stability of the protein molecule. The exact overall contribution of the various weak intramolecular bonds towards the conformational stability is dependent on the amino acid sequence of the protein, which is different from one protein molecule to another. The transition midpoint (T_m) is the temperature where 50% of the protein is in its native conformation and the other 50% is denatured. The T_m of proteins generally represent their relative thermal stability ^[27] The value of the T_m for a protein is a reflection of the strengths of various intramolecular forces that hold the native protein conformation together. In certain proteins, aggregation appear to occur with only minor conformational changes. An example of this is the self-association of insulin; in such case, a technique that detects minor alteration in protein conformation such as circular dichroism (CD) might be more sensitive than measuring the T_m of the protein. When exposed to various stress factors, proteins are prone to denature (alter their native secondary or tertiary structure). These stress factors include temperature, chemical degradation, surface adsorption, formulation conditions such as pH, salts and other additives. ^[28].At elevated temperatures, most proteins unfold and at temperatures below their unfolding transition (T_m), where only partial unfolding is observed, proteins start to aggregate. The resistance of proteins to unfolding, where proteins are considered thermodynamically stable, varies among different proteins and depends on a combination of various forces that contribute to the stability of the protein’s globular conformation. ^[29] In addition to high temperatures, low temperatures below the freezing point can also promote protein aggregation by changing the physical properties of the frozen solutions. In the presence of a polymeric excipient, freezing can easily cause phase separation, promoting protein aggregation. ^[30] In order to reduce aggregation due to thermal instability, protein-based biopharmaceuticals are commonly kept at cold temperatures (2-8 ⁰C) to restrict their conformational flexibility and to preserve their structural integrity. In non-native protein aggregation, the monomeric protein unfold either partially or fully, so that aggregation-sensitive regions, such as hydrophobic residues normally buried in the globular protein core, are exposed, this may then cause the proteins to stick together. ^[31] The rate-limiting step in non-native protein aggregation is associated with unfolding or partial unfolding of the protein from its native conformation. ^[32] Thermally induced protein unfolding is often accompanied by immediate aggregation due to exposure of the hydrophobic residues. Examples include hemoglobin, ^[33] Granulocyte colony-stimulating factor (GCSF), ^[30]recombinant human growth hormone (rhGH), ^[34]andinsulin. ^[35] Chemical degradation resulting in chemical modification that may lead to protein aggregation; it is difficult to predict the effect of a particular structural change on protein aggregation tendency, because the site at which chemical modification occurs may or may not directly participate in the aggregation. For example, oxidation of Meth residue in the variable domain of an IgG light chain induced noticeable secondary and tertiary structural changes and made this protein more sensitive to stirring-induced aggregation. ^[36] In addition, such chemical degradation may reduce the bioactivity of the protein-based biopharmaceutical. Adsorption to a hydrophobic surface could be a cause of protein partial unfolding, which may lead to protein aggregation. Some proteins tend to expose their hydrophobic moieties, normally present in the core of the native protein structure when come in contact with a hydrophobic surface.The adsorbed partially unfolded protein molecules form aggregates, leave the surface, return to the bulk of the aqueous phase, and eventually form larger aggregates that precipitate.This is the proposed mechanism for aggregation of insulin in aqueous media as it comes in contact with hydrophobic surfaces such as plastic syringes. ^[37] Many studies have showed that the magnitude and duration of shear exposure does not cause protein aggregation. ^[38]A probable reason for the frequent association of protein aggregation with processes that exert shear forces on fluids is the concomitant presence of interfaces in high-shear process equipment rather than the stress of shearing. ^[39] Thermodynamic methods such as differential scanning calorimetry (DSC) can be useful in determining the effect of temperature on protein’s conformational stability by measuring T_m.^[40] This technique can be used to select optimal pH, buffer species, stabilizers and other formulation factors that produce the most stable conformational protein. ^[41]Since DSC cannot detect minor alteration in the protein conformation such as α-helical content of a protein; then spectroscopic methods, particularly far-UV (180–260 nm) circular dichroism can be used. ^[31] The colloidal stability of a protein against native protein aggregation depends on the net charge of the protein and the hydrophobicity of the external surface of the globular protein. ^[42] In this aggregation route, the protein molecules retain their normally folded native conformation, but have aggregation-sensitive regions on their surface, such as localized charged regions or hydrophobic moieties that cause the proteins to stick together and aggregate.Such associations can be simply electrostatic or both electrostatic and hydrophobic depending on the experimental conditions . Native protein aggregation often leads to formation of reversible oligomers aggregates that can be considered the precursors of irreversible aggregates precipitates. ^[43] Proteins aggregate at a neutral pH mainly because surface hydrophobicity increases. It has been demonstrated that the surface hydrophobicity of ovalbumin markedly increased as the pH was decreased from 8.0 to pH 7.0. ^[44]A key parameter to measure the tendency of protein–protein self–association due to surface charge is the second virial coefficient (B22). A positive value indicates protein–protein net repulsion while a negative value indicates protein–protein net attraction. In the latter case, protein–protein interactions are favored over protein–solvent interactions, potentially dominating protein aggregation.^[42] The B22 values can predict native protein aggregation due to surface charge only, but not due to surface hydrophobicity. Further, the relative B22 values do not predict aggregation tendency because of conformational stability. ^[45] It has been demonstrated that ovalbumin readily aggregates during incubation at 37°C at pH 4.0, but no aggregation was observed at pH 7.4. However, similar B22 values were obtained in all solution conditions irrespective of the different aggregation tendencies. ^[37] A more useful approach for investigating the effect of native protein-protein interactions due to either surface charge or surface hydrophobicity is to determine the solubility of the candidate protein in the solution of interest. It appears that there is a strong correlation between protein solubility and protein-protein interactions; protein solubility decreases when the protein-protein interactions become less repulsive or more attractive. ^{[46, 47]} The isoelectric point (pl) of a protein is the pH at which its net charge is zero. At a pH below the pI, the protein net charge is positive and negative when the pH is above the pI. The pI of a protein to some extent determines the solubility of a protein at a given pH, with the lowest solubility theoretically occurring at the pH equivalent to the pI. Titration of the pH away from the pI to either more basic or more acidic conditions often improves solubility within the pH limit of protein chemical structure and native conformation retention. ^[48] Each protein has a solubility limit, which is strongly dependent on the solution conditions. Proteins can easily aggregate, precipitate, or crystallize when protein concentration exceeds this limit. ^[49]

Influence of formulation conditions on protein aggregation

A general mechanistic understanding of how formulation conditions affect conformational and colloidal stabilities can help to provide insight into why some conditions succeed and others fail at reducing protein aggregation rate. Although freshly isolated proteins are folded into a distinct three-dimensional globular structure in water, this folded structure is not necessarily retained indefinitely;hence, protein molecules must be stabilized by certain additives and stored under appropriate conditions in order to maintain their stability. ^[50] The effect of four major formulation additives namely, pH and buffering agents, salts, non-ionic surfactants and polyols will be described. These additives can be varied sequentially one at a time or in combinations.

A-pH and buffering agents:

The solution pH can affect conformational/colloidal stability of proteins and changing the solution pH could potentially change the aggregation mechanisms, as pH can affect protein aggregation through one or more of the following three possible mechanisms:

1- pH changes can be simply an effect of altered charge-charge interactions; proteins aggregate at a neutral pH mainly because of protein–protein interactions or surface hydrophobicity.A study on the stability of insulin in the pH range of 3–9 showed that turbidity of insulin at room temperature was fastest at pH 5.6 near Ip 5.5 of insulin in a 10 mM NaCl solution [48]. Another similar study, observed that the aggregation tendency of Granulocyte-colony stimulating factor (GCSF) is significantly higher at pH 6.1 or 7.0 than at pH 3.5. Although GCSF remains conformationally native and compact from pH 2 to 7. B 22 values of the protein changed from positive to negative as the pH was increased from 3.5 to 7.0, indicating that pH-induced protein–protein attractions are likely responsible for the rapid aggregation at neutral pH [16]. Charged species are more easily hydrated than non-charged species and have a greater driving force for interaction with water. The number, density, and location of charged residues on the surface determine solubility and hence exert a profound influence on the stability of proteins. ^[51] However, pH values, which favor negative surface charge, appear to contribute more towards increasing protein’s solubility than positive surface charge. This is best explained by the strong water-binding properties of glutamic and aspartic acid compared to lower water binding ability of lysine or arginine. ^[52]

2- pH-induced protein partial unfolding, results in non-native aggregation. It was found that the aggregation of a monoclonal antibody (mAb) was accelerated at pH 4.0 compared to pH 7.4, apparently due to partial unfolding of the protein at pH 4.0. ^[53]

3-pH change may alter chemical stability and pathways that could lead to protein aggregation. This is illustrated by the direct deamidation involving the hydrolysis of Asparagine and Glutamine side chain amides at acidic pH or deamidation via the succinamide intermediate at more basic pH. ^[54] It was observed that two major aggregation pathways in an IgG2 molecule at pH 4.0 and 6.0 at 37 °C exist. The major aggregation pathway at pH 4.0 is the formation of both dimers and high molecular weight containing cleaved antibody molecules (in the CH₂ domain), while at higher pH 6.0, the major pathway is the formation of a covalent dimer while formation of higher molecular weight aggregates was largely suppressed at this pH. ^[55]From above, it appears that optimization of pH for each protein must be determined based on several instabilities (both chemical and physical) simultaneously. If slight variation in the pH affects the rate of protein aggregation or stabilization, then buffers can be used to maintain the optimal pH. Different buffer systems and/or at different buffer concentrations can significantly affects aggregation behavior of proteins. ^[56] A study on the effects of buffering species on the aggregation of Interferon at pH 7 at 50° C showed that the protein aggregation rate was fastest in phosphate buffer, intermediate in Tris buffer, and slowest in histidine buffer. ^[57]

B-Salts:

At low concentrations, all salts increase the solubility of the protein, this effect is known as salting-in. The salting-in effect is due to the charge screening effects of salts in reducing electrostatic interactions between protein molecules. ^[58] At salt concentration higher than that needed to induce charge screening effect, some salts interact preferentially with the solvent (salting out salts or kosmotropes) while others interact preferentially with the protein (salting in salts or chaotropes). Salting out salts stabilize protein conformations through the preferential exclusion mechanism. ^[59] The preferential exclusion of the salt from the protein surface lead to decrease in the protein’s solubility in the solvent as a result of decreased interaction of the protein with the solvent. The decreased interaction of the protein with the solvent result in a more compact globular protein and increased thermal stability of the protein. The continued increase in the concentration of the salting out salt leads to protein precipitation. Different salts cause precipitation at different concentrations. The ranking in effectiveness of precipitation and stabilization follows the well-known Hofmeister series. ^[60] Kosmotropic stabilization study reported that the aggregation levels of heat-treated low molecular-weight urokinase (LMW-UK) was reduced in the presence of up to 0.19 M ammonium sulfate. Little additional benefit was observed above 0.19 M, and at concentrations above 1.7 M, the LMW-UK protein precipitates. ^[61]

C-Non-ionic surfactants:

Non-ionic surfactants such as tween 80 or tween 20 have been shown to prevent aggregation through improving water solubility of the globular protein, leading to the stabilization of protein conformation. ^[62] Currently tweens are incorporated in several marketed protein-based biopharmaceuticals. ^[63] The mechanism of action of non-ionic surfactants is suggested to be related to the weak binding of their hydrophobic tail to the hydrophobic patches on protein surfaces. Such interaction will partially or completely block the aggregation-prone hydrophobic sites on the protein surface, hence preventing protein-protein interactions. The optimal amount of surfactants required for stabilization, is the amount needed to saturate the hydrophobic patches on the globular protein surface. ^[64]

Non-ionic surfactants were shown to be very effective to protect proteins from shaking/shipping/mixing-induced aggregation. The common surfactants used in protein formulations include tweens. ^[65] It has been shown that tween 20 can bind to recombinant human growth hormone (rhGH), and the binding stoichiometry between the two was found to be 2.5:3.5 (tween 20: rhGH) by electron paramagnetic resonance. ^[62] Such interaction would block or partially block the aggregation-prone hydrophobic sites on the protein surface, preventing protein-protein interactions. Such interactions was also observed to increase the free energy of unfolding, leading to protein stabilization. ^[66]

D-Polyols:

Polyols encompass a class of excipients that include sugars (mannitol, sucrose, trehalose and sorbitol) and other polyhydric alcohols (propylene glycol and glycerol). Polyols interact preferentially with water, and are excluded from protein structure. The non-specific preferentially excluded polyols result in increasing the thermodynamic stability of the native globular protein conformation. ^[67] Polyols protective effect on protein aggregation generally increases with increasing polyol concentration. This has been observed when adding sucrose or sorbitol to bovine serum albumin. Many proteins are stabilized by these compounds, For example, the rate of aggregation of interferon at 50 ° C was reduced gradually with increasing sucrose concentrations at 0.25, 0.5, 0.75, and 1.0 M. ^[68] The thermal stability study of hen egg-white lysozyme (HEWL) at 100 mM, revealed that the melting temperature was significantly increased in a wide pH range of 3.5-9.5 in the presence of 1 M sorbitol, and an increase of as high as 9.5°C was observed at pH 9.5. ^[69] It was proposed that preferential exclusion is a common element in aqueous sugar systems at the concentrations typically utilized, and therefore, it is reasonable to assume that other simple sugars would have similar stabilizing effects as sucrose. ^[70]The resulting thermodynamic equilibrium will favor the native protein state and make it more structurally stable. ^[71]

Predicting Long-Term Storage Stability of Protein-based Biopharmaceuticals:

The challenge to produce protein-based biopharmaceuticals that are stable over long term periods has further increased in recent years due to pressure to move away from lyophilized formulations, which need reconstituting by the patient, toward more convenient liquid formulations, which can be directly injected. As explained in this review, proteins in solution can degrade by means of several mechanisms during extended storage, and a common degradation route is aggregation of the protein over time. Protein-based biopharmaceuticals developers have two main strategies to create liquid formulations with long shelf lives and a resistance to the formation of aggregates; these are the designing of molecules that are aggregation resistance and the inclusion of additives that inhibit the formation of aggregates in liquid formulations. These two options may take a long time to develop and may finally produce an incompetent aggregation resistance product. The ideal way is to develop a rapid method of prediction at the early stage of the product development, with a reasonable level of confidence. As previously stated, there are two pathways by which protein molecules can aggregate. In the first pathway, some of the proteins in solution unfold partially or fully so that aggregation-competent regions, such as hydrophobic residues, are exposed and cause the proteins to stick together, a phenomenon described as “non-native aggregation.” In the second pathway, the protein molecules retain their correctly folded, native conformation, but have aggregation-competent regions on their surface, such as localized charged regions or hydrophobic patches, that cause the proteins to stick together and aggregate. The aggregation rate-limiting steps for these two different pathways are quite different, and, therefore, different experimentally measurable parameters may be potentially useful, depending on which pathway is dominant for a particular molecule or formulation.

Protein melting temperature as a as a predictive tool:

One of the most widely used, experimentally determined parameter for screening molecules and formulations for stability is the protein melting temperature (T_m) which is the temperature at which the protein is observed to unfold. T_mmay be determined experimentally by applying a temperature ramp to the protein in the solvent of interest and identifying the temperature at which the protein unfolds using either calorimetric methods, such as DSC, or spectroscopic methods, such as intrinsic protein fluorescence. ^[72-75] Conceptually, a higher Tm indicates that fewer unfolded protein molecules exists and, therefore, less chance of the formation of non-native aggregates over time. T_m can be a useful prescreening tool to identify particularly conformational stable or unstable molecules, but may not, on its own, be predictive of long-term storage stability for all samples. A study on the potentials and the limitations of the predictive power of T_m reported that a correlation exists between T_m and aggregation during storage at 40 °C for two monoclonal antibodies, but not for a third one. The stability of the third antibody appeared to be predicted by its rate of aggregation when heated to 70 °C. ^[73] It can be concluded that T_m can be a useful prescreening tool to identify particularly conformational stable or unstable molecules, but may not, on its own, be predictive of long-term storage stability for all formulations.

High-temperature aggregation kinetics as a predictive tool:

An additional approach to the use of Tm as a predictor of storage stability is to observe the kinetics of aggregation when the formulation is held at a temperature that accelerates the formation of aggregates. Such measurements essentially explore a combination of the time-dependent rates of unfolding and the aggregation of these proteins. A number of studies has demonstrated that measuring aggregation rates at elevated temperatures for rapid formulation and candidate screening, is important in predicting behavior at lower temperatures and longer times. Protein intrinsic fluorescence technique can be used to monitor the time-dependent rate of thermally induced unfolding of protein, while the corresponding rate of aggregation can be obtained using light scattering technique. ^[76-79]

Protein-protein interactions as a predictive tool:

The attractive interactions between native proteins in solution can potentially lead to the formation of aggregates, and it is therefore essential to measure the strength and nature (attractive or repulsive) of these interactions for a candidate protein-based biopharmaceutical or formulation. The resistance to aggregation due to native protein-protein interactions is referred to as the “colloidal stability” of the protein. One of the most developed method for measuring protein-protein interactions in solution is static light scattering, which requires only the protein concentration-dependent light-scattering intensity from the protein of interest in the specific solution. By combining these data with suitable physical and instrumental constants, one can generate a graph, known as a Debye plot, from which a value called the second virial coefficient (B22) can be obtained. The sign of this value indicates whether the protein-protein interactions are attractive (a negative value) or repulsive (a positive value) while the magnitude of the value indicates the strength of the interaction. ^[80,
81]

Protein solubility as a predictive tool:

An alternative approach to investigate the effect of native protein-protein interactions is to determine the solubility of the candidate protein in the solution of interest. A study based on two strategies to formulate an IgG. One approach sought to stabilize the conformation of the protein, and the other sought to improve its solubility. The effect of the formulations on conformational stability was evaluated with DSC, and the effect on protein solubility was investigated using ammonium sulfate precipitation. All formulations were stored at 4°C for nearly a year, and the rate of aggregate formation was measured to directly assess the effectiveness of the formulation in preventing aggregation during long-term storage. The key result of this study was that, for the molecules and formulations studied, the effect of the formulation on the solubility of the protein, rather than the effect on the conformational stability, was key to improving the long-term aggregation resistance of the protein. ^[82]

Clinical consequences of protein-based biopharmaceuticals:

Protein-based biopharmaceuticals have revolutionized the treatment of many diseases. In the near future, many more protein-based biopharmaceuticals are likely to become available to treat a wide range of diseases. It has been recognized that these proteins may induce humoral and cellular immune responses. The consequences of an immune reaction to a protein-based biopharmaceutical range from transient appearance of antibodies without any clinical significance to severe life threatening complications such as anaphylaxis, neutralization of the effectiveness of lifesaving or highly effective therapies, or neutralization of endogenous proteins with non-redundant functions and decrease in efficacy and induction of autoimmunity, including antibodies to the endogenous form of the protein. Many factors may influence the immunogenicity of protein-based biopharmaceuticals. These include patient and/or product-related factors. Patient-related factors that might predispose to an immune response include underlying disease, genetic background, immune status, including immunomodulation therapy. Product-related factors also influence the likelihood of an immune response, e.g. intensity of treatment, route of administration, source of protein (human or nonhuman), manufacturing process (impurity profile, contaminants), formulation and stability characteristics (degradation products, aggregates) of a given protein and dose, dosing interval and duration of treatment. ^[83] A review article has indicated that all exogenous proteins, including protein-based biopharmaceuticals, have the potential to cause antibody formation. The reported incidence of antibody formation with protein-based biopharmaceuticals varies widely between proteins and between studies (depending on the assay techniques used). The clinical consequences of antibody formation vary with the type of antibody present; for example, neutralizing antibodies are more likely to cause loss of efficacy than non-neutralizing antibodies. Manufacturing, handling, and improper storage can introduce contaminants, or alter the 3-dimensional structure of the protein via oxidation or aggregate formation. Various strategies s have been suggested where protein-based biopharmaceuticals might be modified to reduce their immunogenicity, including PEGylation, site-specific mutagenesis, exon shuffling, and humanization of monoclonal antibodies. In the future, it may even be possible to predict the immunogenicity of new protein-based biopharmaceuticals more accurately, using specifically designed animal models, including nonhuman primates and transgenic mice. ^[84]The biopharmaceutical concerns of protein-based biopharmaceuticals such as, poor shelf life, rapid degradation in the physiological environment, poor solubility, immunogenicity and antigenicity, can be overcome by utilizing the beneficial properties of polyethylene glycols and PEGylation. ‘PEGylation’ is the process of chemical attachment of PEG to bioactive proteins and peptides, to modify their pharmacokinetic and pharmacodynamics properties. Therefore, PEGylation can be applied to modify the physicochemical properties of the parent drug either free form or encapsulated form to improve its immunogenicity, cellular uptake, spatial placement and biological activity. The scope of PEGylation in the near future is still intensifying, as it is the most reliable tool available to help the innovative medicinal products meet the clinical and regulatory standards. ^[85] Heat Shock Proteins (HSPs) constitute a group of proteins that play a crucial role in the process of protein folding. HSPs are known to modulate a number of key apoptotic factors. High expression of these proteins is reported in an array of cancers, such as breast, prostate, colorectal, lung, ovarian, gastric, oral and esophageal cancer. Ample amount of investigations were carried out on a variety of cancers suggesting HSPs as a promising hallmark in cancers. Their expression profile in several tumors elucidates that they help in proliferation, invasion, metastasis and death of cancerous cells. Detection of the levels of heat shock proteins and their specific antibodies in the sera of diseased individuals can play an important role in cancer diagnosis. ^[86]

Fusion proteins are products of gene fusions resulting from chromosomal rearrangements that are capable of acting as potent oncogenes. They operate via a myriad of mechanisms, including kinase activation or the deletion of regulatory microRNAs. ^{[87, 88]} Their clinical relevance is evident in the insight they provide into early tumorigenesis as well as their role in facilitating the development of novel targeted therapies and diagnostic applications. Gene fusions were first identified in hematological malignancies with the initial description of BCR-ABL in chronic myeloid leukemia in 1960.^[89] Discovery of this fusion led to the development of targeted first-line therapies such as the tyrosine kinase inhibitor Dasatinib. ^[90] Various events are capable of generating oncogenic fusion proteins, including inter-chromosomal translocations or intra-chromosomal rearrangements such as translocations, deletions, and inversions. ^[91] Interestingly, viruses are also capable of generating fusions harboring oncogenic activity. ^[92] Fusion proteins are being recognized for their relevance in head and neck neoplasms and have thus far been identified in malignancies originating from the nasopharynx, salivary glands, maxilla, auditory meatus, lacrimal glands, thyroid, esophagus, and midline head and neck structures.^[93-95] They provide logical targets for therapies that may benefit patients with unresectable head and neck malignancies, while providing valuable contributions to our understanding of their pathogenesis, diagnosis, and prognosis. ^[96] It has been reported that sonication of a range of structurally diverse proteins results in the formation of aggregates that have similarities to amyloid aggregates. The formation of amyloid is associated with, and has been suggested in causing wide range of protein conformational disorders including Alzheimer’s, Huntington’s, Parkinson’s and prion diseases. Ultrastructural analysis by electron microscopy reveals a range of morphologies for the sonication-induced aggregates, including fibrils with several nanometers dimensions. The addition of preformed aggregates to un-sonicated protein solutions results in accelerated and enhanced formation of additional aggregates upon heating. These results have important implications for the use of sonication in food, biotechnological and medical applications, and for research on protein aggregation and conformational disorders. ^[97]

CONCLUSION:

Long-term storage stability against aggregation of protein-based biopharmaceuticals can be achieved through understanding of protein aggregation pathways and how excipients interact with proteins. If protein aggregation was due to conformational instability then increasing thermodynamic stability of the native state as conferred by protein stabilizers, in addition to optimum pH and salt type and concentration would help in controlling aggregation caused by conformational instability of the tertiary structure of the protein. Aggregation of native conformations or colloidal instability of the protein can be reduced by decreasing intermolecular hydrophobic interactions between the native conformation using aggregation suppressors such as non-ionic surfactants or very low concentration of simple salts such as NaCl. The use of anti-adhesion agents such as serum albumin or non-ionic surfactants would help in controlling aggregation caused by surface adsorption. Currently, no single measurement can be considered predictive of long-term storage stability for all protein-based biopharmaceuticals in all formulations. To improve the predictive capabilities of early-stage protein-based biopharmaceuticals candidate and formulation screening, multiple measurements are needed to probe different potential pathways to aggregation. These measurements can be used to investigate the dominant mechanisms that lead to aggregation for a particular molecule, so that strategies to alleviate these may be rationally designed. An essential consequence of protein aggregation is their possible contribution in reducing efficacy and inducing immunogenicity, which make stability assessment a fundamental quality control attribute for protein-based biopharmaceuticals. Immune responses to protein-based biopharmaceuticals are usually only of clinical significance if they are associated with the development of treatment resistance. Although various means to reduce the immunogenicity of protein-based biopharmaceuticals have been suggested, monitoring for antibodies during clinical trials and post-marketing surveillance remains an important issue for all protein-based biopharmaceuticals.

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Received on 21.11.2019 Modified on 18.12.2019

Research J. Pharm. and Tech 2020; 13(9):4443-4452.

DOI: 10.5958/0974-360X.2020.00785.4